Pseudomonas aeruginosa is ubiquitous, opportunistic pathogen that primarily infects immune-compromised individuals, including AIDS and transplant patients, severe burn patients, and those with cystic fibrosis (CF). In the context of CF, P. aeruginosa establishes a chronic condition whose morbidity and mortality results from lung damage. Due to the uncanny antibiotic resistance, CF patients infected with Pseudomonas often have chronic infections with limited therapeutic options. Therefore, for improved efficacy in treatment, a basic understanding of the pathogenic mechanisms utilized by this organism needs to be examined as possible therapeutic targets. While a majority of previous studies have focused on the initial stages of colonization and infection, we propose a new approach in searching for treatments. Mounting evidence indicates that microaerophilic metabolism and a biofilm mode of growth may be involved in P. aeruginosa pathogenesis; however, explanations for a mechanism have yet to be discussed. One virulence factor produced by P. aeruginosa under these growth conditions is HCN. Micromolar amounts of HCN inhibit the respiratory electron transport chain and several metalloenzymes (e.g., catalase, peroxidase, superoxide dismutase) of eukaryotic cells. We have discovered that AlgR, a regulator of the virulence factor, alginate, also activates HCN production in mucoid P. aeruginosa. Using the Pseudomonas Affymetrix GeneChip and S1 nuclease protection assays, we demonstrate that AlgR is controlling hcnA, encoding hydrogen cyanide synthase. Moreover, direct measurement of HCN production revealed that mucoid P. aeruginosa produce up to 2.5 mM of HCN in 4 h. Our preliminary data indicate two new roles for AlgR: i) AlgR controls HCN production and ii) AlgR is able to switch from a repressor in nonmcoid P. aeruginosa to an activator in mucoid bacteria on the hcnA promoter. Additionally, we demonstrate that AlgZ/FimS is playing a role in this process. The hypothesis to be tested is: AlgR activates HCN production in mucoid P. aeruginosa. We will test this hypothesis with four specific aims: i) we will determine the requirements for AlgR protein-DNA interaction within the hcnA promoter; ii) we will determine if phosphorylation is required for AlgR activation of hcnA expression in mucoid P. aeruginosa; iii) we will determine the amount of HCN production and hcnA expression within biofilms, and; iv) we will determine the effect of HCN production on lung epithelial and human neutrophil cells in vitro. Thus, at the end of our proposed studies, we hope to elucidate new possible therapeutic target as well as gaining a better understanding of Pseudomonas biology and pathogenesis.